Design and synthesis of close analogs of LCRF-0004, a potent and selective RON receptor tyrosine kinase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2015.04.056

Abstract New carboxamide head group analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Potent and selective inhibitors of RON enzyme versus c-Met RTK were obtained.

Full text of DOI:10.1016/j.bmcl.2015.04.056

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of close analogs of LCRF-0004, a potent
and selective RON receptor tyrosine kinase inhibitor
⇑
Stéphane L. Raeppel , Franck Raeppel, Eric Therrien
ChemRF Laboratories, 3194 rue Claude-Jodoin, Montréal, QC H1Y 3M2, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
New carboxamide head group analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were
designed and synthesized. Potent and selective inhibitors of RON enzyme versus c-Met RTK were
obtained.
Received 6 March 2015
Revised 14 April 2015
Accepted 17 April 2015
Available online xxxx
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
RON
c-Met
RTK inhibitors
Head group
Thieno[3,2-b]pyridine scaffold
Oncology
N
N
The receptor tyrosine kinase RON (Récepteur d’Origine Nantais,
also known as human MST1R, for macrophage stimulating 1 recep-
tor) is the cell surface and transmembrane receptor with a cyto-
plasmic tyrosine kinase domain for its extracellular endogenous
ligand MSP (for macrophage stimulating protein, also known as
HGFL, for hepatocyte growth factor-like). RON belongs to the
MET proto-oncogene family and shares significant structural and
functional homology with c-Met RTK, and is normally expressed
at low level in epithelial cells. The MSP/RON signaling pathway is
involved in several important biological processes, including
macrophage activity, wound healing, and epithelial cell behavior.
Deregulation of RON has been described in numerous types of
cancers1 including colorectal,2 breast,3 lung,4 pancreas,5 prostate6
and bladder and occurs mainly through wild type receptor overex-
pression or expression of variants harboring different deletions
within the extracellular domain, leading to constitutive receptor
activation.
Metastases in the body are the main cause of death for cancer
patients. A recent study has further shown that MSP/RON axis
facilitates tumor metastasis by suppressing host antitumor immu-
nity.7 The MSP/RON signaling pathway favors the conversion of
micrometastatic lesions to overt metastases by suppressing antitu-
mor immune responses. However, the loss of RON functions in the
host promotes an effective antitumor CD8+ T-cell (lymphocytes)
H
N
1
F
5
3
CF3
O
O
N
S
LCRF-0004
N
N
A
B
C
Figure 1. Structure of LCRF-0004.
response, hence inhibiting the outgrowth of metastatic cancer
cells. Consequently, RON inhibitors may potentially prevent the
outgrowth of micrometastases in cancer patients. A new study
has reported an epigenetic reprogramming pathway that is
required for breast cancer metastasis.8 Concerted differential
DNA methylation is initiated by the activation of the RON receptor
tyrosine kinase by its ligand MSP. Analysis of human breast cancers
revealed that this epigenetic program is significantly associated
with poor clinical outcome. Furthermore, inhibition of RON kinase
activity with a pharmacological agent blocks metastasis of patient-
derived breast tumor grafts in vivo.
As part of our internal research program focusing on the
discovery of novel agents to fight and/or control the proliferation
of metastatic cancers and metastasis, preliminary and new data
were collected using LCRF-0004 (Fig. 1) known to be a potent
⇑
Corresponding author. Tel.: +1 514 244 1681.
E-mail address: stephane.raeppel@gmail.com (S.L. Raeppel).
http://dx.doi.org/10.1016/j.bmcl.2015.04.056
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Raeppel, S. L.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.056

2
S. L. Raeppel et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
(MPM, H226 cancer cell line).11 Asbestos exposure is the principal
etiological agent of MPM cancer type.12
Table 1
Enzymatic and cellular inhibitory activities of LCRF-0004 and close analogs
Deprived of structural information about the binding of LCRF-
0004 within the RON enzyme, we deemed important to synthesize
and evaluate close analogs of LCRF-0004 to gain insights about this
inhibitor and establish structure activity relationships (SAR). As a
first step, we decided to screen various head groups (Fig. 1, Part
C), as this section of the molecule is believed to be the key motif
for the observed selectivity over c-Met. Herein, we describe our
efforts in the synthesis of small molecules, close analogs of LCRF-
0004, as potent and selective inhibitors of RON tyrosine kinase
versus c-Met RTK.
F
HG
O
N
S
N
N
Compds Head Group (HG)
RON (h)
kinase IC50
M)
Phospho-c-Met in
MKN-45 IC50 M)
(
l
(l
The synthesis of new analogs is described as follows: 1-phenyl-
2-(trifluoromethyl)-1H-pyrrole-3-carboxylic acid 4 was prepared
in 4 steps from ethyl 2-diazoacetate via ethyl 5-ethoxy-2-(trifluo-
romethyl)-4,5-dihydrofuran-3-carboxylate 2.13 Amide coupling
between intermediates 4 and 514 gave access to compound 6 with
a pyrrole head group (Scheme 1). 1-Phenyl-5-(trifluoromethyl)-
1H-imidazole-4-carboxylic acid 9 was prepared in 3 steps from
aniline via ethyl 1-phenyl-5-(trifluoromethyl)-1H-imidazole-4-
carboxylate 8.15 Amide coupling between intermediates 9 and
N
N
H
LCRF-
0004
N
0.012
6.87
CF3
N
O
O
O
H
N
6
0.010
0.013
n.a.
CF3
N
N
N
H
N
10
2.40*
5
afforded compound 10 with an imidazole head group
CF3
N
N
(Scheme 2). 1-Phenyl-5-(trifluoromethyl)-1H-1,2,3-triazole-4-
carboxylic acid 12 was prepared in 2 steps from phenylboronic
acid via ethyl 1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazole-4-
carboxylate 11.16 Amide coupling between intermediates 12 and
5 provided compound 13 with a triazole head group17 (Scheme 3).
1-Phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid 15
was prepared in 2 steps from ethyl 5-(trifluoromethyl)-1H-pyrazole-
4-carboxylate via ethyl 1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylate 14.18 Amide coupling between intermediates 15 and 5
gave rise to compound 16 having a pyrazole isomer head group
(Scheme 4). Amide coupling between 1-(4-fluorophenyl)-5-(triflu-
oromethyl)-1H-pyrazole-4-carboxylic acid and 5 afforded com-
pound 17 with a para-substituted phenyl head group (Scheme 5).
Amide methylation of LCRF-0004 provided compound 18 with a
tertiary amide linker connecting the head group to the central phe-
nyl ring (Scheme 5).
Our design was guided by molecular docking studies using an
X-ray crystal structure of c-Met kinase domain (PDB: 3U6I)19
which shares a high sequence homology with RON. The docking
of LCRF-0004 using the Fitted program20 revealed a class II
extended conformation with the head group (Part C) deeply
engaged in the hydrophobic back pocket of the enzyme interacting
with the backbone NH of Asp1222. A possible intramolecular
hydrogen bond between one fluorine atom from the trifluo-
romethyl group and the NH-acidic carboxamide of LCRF-0004
may occur to form a 7-membered ring. The thieno[3,2-b]pyridine
scaffold (Part B) interacts with the hinge binding domain (NH of
Met1160) and 1-methyl-1H-imidazole substituent (Part A) is posi-
tioned in the solvent-exposed area (Fig. 2).
H
N
13
16
0.017
0.095
0.67
CF3
O
3.77*
N
N
H
N
CF3
O
O
O
N
N
H
N
4.37*
n.a.
F
17
18
0.044
0.580
CF3
N
N
N
CF3
*
‘Plateau’ was observed; n.a. (data not available).
and selective inhibitor of RON receptor tyrosine kinase, reported by
MethylGene.9 Low nanomolar biological activity of LCRF-0004
was determined (IC50 = 12 nM) in a RON kinase domain enzymatic
assay (Table 1).10 Furthermore, the selectivity of LCRF-0004
for RON over c-Met was confirmed in c-Met- and RON-driven
cellular assays. High micromolar inhibitory activities were
observed on c-Met-phosphorylation in MKN-45 (gastric carcinoma,
IC50 = 6.87
l
M) cell line. On the other hand, LCRF-0004 potently
As previously disclosed, the presence of trifluoromethyl and
phenyl substituents on the pyrazole head group of LCRF-0004 is
essential for the inhibition of RON activity.9 In the present study,
we demonstrated that it is possible to replace the pyrazole head
group of LCRF-0004 by other five-membered heterocycles such
as pyrrole (6), imidazole (10), and triazole (13) without loss of
activity for the RON enzyme inhibition. These new analogs are
equipotent when compared to LCRF-0004, paving the way for
more innovation (Table 1). On the other hand, some activity for
c-Met starts to appear in MKN-45 cell-based assay in which c-
Met RTK is overexpressed (e.g. compound 13 versus LCRF-0004).
However, moving the phenyl substituent on the adjacent nitrogen
of the pyrazole ring of LCRF-0004 such as in compound 16 is detri-
mental for the RON inhibition. The three substituents on the pyra-
zole head group shall be contiguous in order to respect the 5-atom
inhibited the RON-phosphorylation in the low nanomolar range
in PC-3 (prostate cancer, IC50 = 30-40 nM)9 and HT-29 (colon can-
cer, IC50 = 30 nM)10 cancer cell lines where RON is overexpressed.
These results demonstrate that this compound is selective for
RON RTK versus c-Met RTK and penetrates into cancer cells.
Moreover, LCRF-0004 showed potent antiproliferative activities
against HCT116 (colon carcinoma, IC50 = 0.08
(breast adenocarcinoma, IC50 = 0.10 M), and A549 (lung cancer,
IC50 = 0.10
M) cancer cell lines using MTT assays,10 but we cur-
rently do not know by what exact mechanism takes place this bio-
logical effect. Through an academic collaboration, more advanced
studies were conducted which further confirmed that targeting
the kinase domain of the RON receptor with LCRF-0004 is an effec-
tive interventional strategy in malignant pleural mesothelioma
lM), MDAMB-231
l
l
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S. L. Raeppel et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
F3C
O
CF3
O
NH2
O
O
Rh2(OAc)4
hydroquinone cat.
O
O
O
O
pyridine
N2
F3C
OEt
O
100oC
EtO
OEt
DCM
0oC
AcOH
100oC
N2
CF3
O
1
2
F
NH2
O
5
N
S
4M NaOH
in water
N
N
N
N
HO
EtO
HATU/DIPEA
DMF
MeOH/THF
80oC
CF3
CF3
O
O
70oC
4
3
H
N
N
F
CF3
O
O
N
N
S
6
N
Scheme 1. Synthesis of compound 6.
O
CN
OEt
TFA/PPh3
Et3N
1N NaOH
in water
Cl
CF3
N
NH2
NaH
N
EtO
N
CCl4
MeOH/THF
50oC
THF
CF3
O
0oC to reflux
0oC
7
8
5
N
H
N
F
N
HATU
N
DIPEA
N
CF3
O
HO
O
DMF
rt
CF3
O
N
S
10
9
N
N
Scheme 2. Synthesis of compound 10.
1) NaN3, Cu(OAc)2
DMSO/water, rt
1N NaOH
in water
N
N
B(OH)2
N
EtO
O
O
MeOH/THF
rt
CF3
11
O
2)
F3C
OEt
piperidine cat.
rt to 80oC
5
N
N
H
N
N
F
HATU
N
N
N
DIPEA
CF3
O
HO
O
DMF
rt
CF3
12
O
N
S
13
N
N
Scheme 3. Synthesis of compound 13.
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4
S. L. Raeppel et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
NHMe
I
NHMe
4M NaOH
in water
N
N
N
NH
EtO
EtO
MeOH/THF
rt
CuI/K2CO3
toluene
reflux
CF3
14
CF3
O
O
5
N
H
N
N
F
HATU
N
N
DIPEA
CF3
O
HO
O
DMF
rt
CF3
N
S
O
16
N
15
N
Scheme 4. Synthesis of compound 16.
N
5
H
N
N
F
F
HATU
N
N
DIPEA
CF3
O
HO
F
O
DMF
CF3
O
N
S
rt
17
N
N
N
N
F
N
MeI
NaH
CF3
O
LCRF-0004
O
DMF
N
S
0oC to rt
18
N
N
Scheme 5. Synthesis of compounds 17 and 18.
Figure 2. Docking pose of LCRF-0004 in c-Met kinase domain.
linker characteristic21 (Fig. 1). Likewise; introduction of a small
substituent (fluorine) in para position of the phenyl ring such as
in compound 17 has also a slight negative impact. The same trend
was observed in a similar series with a different Part A. This effect
was less pronounced with a substituent in ortho or meta posi-
tions.10 Finally, introduction of a tertiary amide linker such as in
compound 18 has a dramatic effect on the RON inhibition and this
compound is 48-fold less active when compared to LCRF-0004. The
presence at this position of a hydrogen bond donor (NH secondary
amide bond) seems to be essential for the activity. Thus, compound
18 cannot adopt an ‘ideal’ bioactive conformation as LCRF-0004
and/or very close analogs (e.g. compounds 6, 10 and 13) are able
to do so, when bound in the catalytic kinase domain of RON.
In conclusion, we have designed and synthesized new potent
inhibitors of RON based on LCRF-0004. Furthermore, this new
search allowed us to study the effect of various head groups on
the RON activity and selectivity over c-Met. Based on these results,
we were able to design and synthesize two new series of potent
RON inhibitors that will be disclosed in due course. Future work
will explore parts A and B of LCRF-0004 (Fig. 1) which should allow
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S. L. Raeppel et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
12. Nowak, A. K. Ann. Cardiothorac. Surg. 2012, 1, 508.
13. Compound was prepared according to Hoffmann, M. G.; Wenkert, E.
Tetrahedron 1993, 49, 1057.
14. Compound 5 was prepared according to WO 2006/010264 A1.
us to further modulate and optimize the in vitro and in vivo activ-
ities and DMPK profile of our inhibitors. More studies from our aca-
demic collaborators using LCRF-0004 as a pharmacological tool for
the drug target validation of RON will be also disclosed soon.
2
15. Compound 8 was prepared according to Huang, W. S.; Yuan, C. Y.; Wang, Z. Q. J.
Fluorine Chem. 1995, 74, 279. During this study we have also synthesized
compound 19.
Supplementary data
N
H
N
CF3
F
N
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.04.
056.
O
O
MeO
MeO
19
lM; Phospho-c-Met in MKN-45:
References and notes
N
Compd 19: RON (h) kinase: IC50 = 0.016
IC50 = 1.06 lM.
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N
N
H
N
F
N
CF3
O
O
MeO
MeO
20
N
.
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Please cite this article in press as: Raeppel, S. L.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.056